Acute disseminated inflammation caused by infection is associated with the elaboration of multiple mediators that act synergistically to amplify organ injury. The objective of this research proposal is to elucidate the intracellular mechanisms responsible for synergistic responses of lung cells to stimulation by more than one inflammatory mediator, events referred to as priming (during which time there is a minimal or no functional response) and primed stimulation (during which time there is marked amplification of a functional response). To facilitate our objective, we have developed models of priming of isolated perfused rabbit lung (IPL), alveolar macrophages, and endothelial cells for enhanced synthesis of eicosanoids, particularly thromboxane (Tx). Preliminary data suggest that priming may induce a sift in the potential of IPL, alveolar macrophages, or pulmonary endothelial cells to synthesize bioactive eicosanoids. We will test this hypothesis by simultaneously quantifying eicosanoids produced following stimulation of primed vs. unprimed IPL and lung cells using state-of-the-art negative-ion, chemical- ionization, gas chromatography/mass spectroscopy and radioimmunoassay. Our findings also indicate that transciptional and translational events are required for bacterial lipopolysaccharide (LPS) priming for enhanced Tx synthesis. We postulate that: (a) LPS priming rapidly increases expression of genes controlling eicosanoid synthesis in lung cells, (b) a specific homologue gene coding for Cox is preferentially expressed during LPS priming, (c) cell-type-specific expression of genes controlling eicosanoid synthesis is induced by LPS priming, and (c) the microvascular endothelial cell is one cell-type responsible for the amplified synthesis of Tx in LPS- primed IPL. Quantitative polymerase chain reactions (PCR), Northern analysis, and in situ hybridization will be used to evaluate MRNAS of enzymes that control eicosanoid synthesis; immunoprecipitation, immunoblot, immunohistochemistry, and biochemical assays will be used to evaluate specific proteins. There is considerable support for the concept that LPS acts in part by first inducing the expression of genes coding for TNFalpha and IL-1. We postulate that LPS priming of IPL may depend on synthesis of one or both of these cytokines. To test this concept we will determine whether inhibitors of IL-1 and TNFalpha block LPS-priming of IPL, and whether nascent MRNAS and cytokines increase in IPL perfusate and lung tissue during LPS priming. These studies of amplified production of inflammatory mediators by the process of priming may clarify the pathogenesis of the multiorgan failure syndrome associated with severe inflammation.